Proteins that misfold in the endoplasmic reticulum (ER) must be degraded efficiently because the accumulation of aberrant proteins can have severe deleterious effects, as evidenced by the large number of protein-misfolding associated diseases. In eukaryotic cells, many proteins that misfold in the endoplasmic reticulum are transported back to the cytosol for degradation. This process is known as ER-associated degradation (ERAD). In addition to its important role in cellular maintenance, the pathway has also been implicated in a number of human diseases, included cystic fibrosis, HIV, and cytomegalovirus. The pathway is only poorly understood, and perhaps one of the most pressing questions about ERAD is how luminal substrates are made accessible to the cytosolic degradation machinery. This proposal is aimed at elucidating the mechanisms of retro-translocation of luminal substrates and identifying the putative protein-conducting channel by investigating the Hrd1 membrane core complex from a yeast model of ERAD. This ER membrane complex contains all of the key ER-membrane inserted components central to to the degradation of luminally misfolded ERAD substrates and is therefore expected to contain the putative retro-translocation channel. By investigating the interactions of the complex with substrate through immunoprecipitation and cross-linking assays, the components that form the channel will be identified. Furthermore, the architecture of the purified Hrdlp core complex and the conformation of the channel will be investigated using electron cryomicroscopy. Identifying the retro-translocation channel will be a key development in understanding the mechanisms of ERAD, the role of the pathway in human diseases, and how retro-translocation machinery is hijacked by a number of viruses and toxins. Relevance: Cells possess special machinery to breakdown improperly assembled proteins, but when this system malfunctions, the accumulation of aberrant proteins can lead to a number of diseases. Dissecting the molecular level details of how this assembly functions in degradation will be important for understanding how breakdowns in the system can lead to disease and will be essential for explaining how certain viruses and toxins can take advantage of and hijack this important cellular machinery.